PROJECT 1 ABSTRACT Project 1 will develop combination photodynamic therapy (cPDT) for non-melanoma skin cancers (NMSC), including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). NMSC are currently treated by surgical excision, but better alternatives are needed, especially for organ transplant patients who develop frequent and aggressive SCC, and for Basal Cell Nevus Syndrome patients who develop hundreds of BCCs. Previously, we discovered that biochemical manipulation with differentiating agents (methotrexate, vitamin D [Vit D], and 5-fluorouracil [5-FU]) enhances heme pathway activity and protoporphyrin IX (PpIX) accumulation. Those findings will now be translated into improved efficacy in the clinic. AIM 1 will examine cPDT for SCC (the most lethal NMSC) using topical 5-FU prior to ALA and blue light. This is a clinical trial to test whether the combination improves efficacy for SCC in-situ in patients at high risk for aggressive NMSC (patients with organ transplants, or highly photodamaged skin). Skin biopsies and blood will be saved for use in Aim 3. AIM 2 will investigate cPDT for BCC (the most prevalent of all cancers). PDT is hampered by a nonhomogeneous photosensitizer (PpIX) distribution in tumors, but Vit D pretreatment induces a more homogenous distribution. Patients with BCC will be pretreated with 5-FU, Vit D, or placebo followed by ALA for 4 hr. A skin biopsy will be taken to assess PpIX distribution; tumors will then be treated with red light. Histological clearance of tumors at 3 months will be confirmed by surgical excision. AIM 3 will investigate new cPDT approaches and new assays to help individualized patient treatments. Liposomal targeting nanoconstructs from Project 3 will be used to deliver small molecule inhibitors against EGFR or VEGF/c-Met (along with photosensitizer BPD) in preclinical models of BCC skin cancer, including a BCC skin explant model from Ptch1 +/- mice. The predictive value of noninvasive monitoring of PpIX in patient skin (using optical tools developed in Project 4), and of measuring p53 levels and thymidine synthase gene alleles, will be tested by correlation with lesion clearance rates in the clinical studies. The preclinical studies will improve understanding of mechanisms and suggest future approaches to individualize the treatments and maximize therapeutic responses to cPDT.